Our overall objective is to understand the regulation of lamellar body (LB) exocytosis in type 2 cells of the intact pulmonary alveolus. We will determine the role of the cytosolic Ca2+ (Ca2+cyt) and mitochondria as determinants of inflation-induced LB exocytosis in normal and LPS-treated lungs. The specific aims are to quantify for the first time, regulation of LB exocytosis by inflation-induced Ca2+cyt oscillations. We will test two hypotheses: First (Specific Aim 1), that Ca2+cyt oscillations increase mitochondrial Ca2+ (Ca2+mit) that increase production of mitochondrial reactive oxygen species (mitoROS), which cause the exocytosis. In Specific Aim 2, we will test the hypothesis that in LPS-treated lungs, NO-induced instability of mitochondrial potential decreases mitoROS production, hence inhibiting LB exocytosis. Procedures: (1) Ca2+ quantification. Fluorometric imaging of Ca2+-sensitive dyes and the Ca2+ FRET probe, Cameleon will be conducted intravitally in single alveoli of the isolated, blood-perfused rat lung, using both wide-angle and optical-sectioning microscopy using methods that have been developed previously. (2) Immunoimaging. Type I and type II cells will be identified by imaging cell-specific immunofluorescence. (3) LB exocytosis. Type II cell exocytosis will be determined in single cells by the loss of cell fluorescence of the acidotropic LysoTracker dyes. (4) NO and ROS. Alveoli will be loaded with the dyes, DAF-2 and DCFH-DA for detection of NO and ROS, respectively. ROS will also be quantified through alveolar expression of the HSP- FRET probe. The hypothesis will be tested through a combination of pharmacological and genetic approaches that interfere with specific signaling intermediates attributable ligation of the P2Y2 receptor. Significance: This proposal addresses a new understanding of surfactant secretion that is critical to lung function, but remains inadequately understood. It is important to know the role of Ca2+ in surfactant secretion, because dysregulation of alveolar Ca2+ may be common to many mechanisms that affect type II cell function and thereby, promote lung injury. Sustained Ca2+ increases may constitute a potent signal for gene transcription and consequently, lung remodeling. If preliminary data bear out, this research will prove for the first time that mitochondrial signaling increase alveolar ROS. New understanding will be achieved regarding dysfunctional surfactant secretion in sepsis. No previous understanding of these mechanisms exists. These proposed studies are therefore, outstandingly novel and important. Project Narrative: This project is to determine fundamental mechanisms underlying alveolar surfactant secretion in normal and septic lungs. The findings of this research are likely to impact understanding of mechanisms of acute lung injury and the development of relevant therapy. [unreadable] [unreadable] [unreadable] [unreadable]